Applications of volumetric imaging can be found in all sectors of society from healthcare, to manufacturing, basic research, and defence. More often than not, current technology limits the acquisition volume and resolution that can be captured in a given time frame. Taking the example of optical microscopy, confocal scanning was until recently the gold standard, yet its acquisition time is limited by the scanning speed which in turn is limited by the laser power and the damage threshold of the sample. Various forms of light sheet microscopy such as orthogonal-plane optical sectioning, selective plane illumination microscopy (SPIM), uitramicroscopy, or digital scanned laser sheet microscopy (DSLM), address this issue. Unwanted background signal and photo-damage is prevented by illuminating the to-be-imaged volume in a stepwise fashion while rapidly capturing as much information as possible from the illuminated parts, e.g. using an adequately placed detector array such as a charge-coupled device camera (CCD). With its various implementations, this technique enables rapid high contrast four-dimensional optical sectioning, and has already revolutionised the study of live organisms.
A problem with conventional light sheet microscopy, based on Gaussian beams, is that high isotropic resolution demands a tightly focussed light sheet and therefore illumination with a high numerical aperture (NA). However, this restricts the distance over which a narrowly focussed light sheet can be maintained, thereby limiting the usable field-of-view. The ability to efficiently image large volumes with a single scan of a standard Gaussian light sheet is therefore incompatible with high resolution imaging.
Imaging large volumes at maximal resolution is key to many areas of research such as imaging for biological studies, whether this be archaebacteria, prokaryotes or eukaryotes and at the subcellular, cellular, tissue and in the whole organism level. Examples include embryology, cell-fate mapping both in stem cells studies and in developmental biology, neurobiology, cell spheroids. High resolution imaging of large volumes may also be used in the area of colloidal physics, and for imaging nanostructures such as three-dimensional meta-materials.
Several solutions have been proposed. Generally these are either restricted to two-photon excitation or require a larger number of sample exposures with the associated consequences for imaging speed and photo-damage that may hamper repeated scans. Bessel beams have been used to extend the imaging volume in light sheet microscopy. The transverse intensity profile of a zeroth Bessel order beam has a central spot and a series of concentric rings away from the beam centre. These rings significantly deteriorate the axial resolution.
Confocal detection of the Bessel beam core can improve the axial resolution. However, its advantage over regular confocal microscopy is small since a significant fraction of the light is rejected by the confocal detection of the light sheet beam, and the scanning speed is limited by the camera because images are acquired line-by-line instead of plane-by-plane. Whilst previous work has shown that the scan volume can be extended by using propagation-invariant, non-diffracting Bessel beams, for single photon excitation the trade-off is a significant loss in signal to noise ratio and resolving power achievable at irradiation levels compatible with biological imaging.